Resonance energy transfer assay with cleavage sequence and spacer

ABSTRACT

Methods for the identification of inhibitors of botulinum neurotoxins are described. Cells are provided that are genetically engineered to express peptides that act as a substrate for a botulinum neurotoxin and the provide reporting groups. Spacer sequences between the reporting groups serve to optimize energy transfer between the reporting groups. Characterization of the energy transfer-dependent signal prior to and following exposure to a botulinum neurotoxin in the presence of a candidate inhibitor provides a measure of the effectiveness of the candidate inhibitor.

This application is a continuation of U.S. patent application Ser. No.15/064,379, filed Mar. 8, 2016, U.S. patent application Ser. No.12/059,570, filed Mar. 31, 2008, which is granted as U.S. Pat. No.9,303,284), which claims priority to U.S. Provisional Application No.60/972,673, filed Sep. 14, 2007, all of which are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The field of the invention is fluorescence resonance energy transfer forprotease assays related to Botulinum toxins and tetanus toxins.

BACKGROUND

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum andare the most potent toxins known. These toxins are a well-recognizedsource of food poisoning, often resulting in serious harm or even deathof the victims. There are seven structurally related botulinumneurotoxins or serotypes (BoNT/A-G), each of which is composed of aheavy chain (.about.100 KD) and a light chain (.about.50 KD). The heavychain mediates toxin entry into a target cell through receptor-mediatedendocytosis. Once internalized, the light chain is translocated from theendosomal vesicle lumen into the cytosol, and acts as a zinc-dependentprotease to cleave proteins that mediate vesicle-target membrane fusion(“substrate proteins”).

These BoNT substrate proteins include plasma membrane protein syntaxin,peripheral membrane protein SNAP-25, and a vesicle membrane proteinsynaptobrevin (Syb). These proteins are collectively referred to as theSNARE (soluble N-ethylmaleimide-sensitive factor attachment proteinreceptor) proteins. Cleavage of SNARE proteins blocks vesicle fusionwith plasma membrane and abolishes neurotransmitter release atneuromuscular junction. Among the SNARE proteins, syntaxin and SNAP-25usually reside on the target membrane and are thus referred to ast-SNAREs, while synaptobrevin is found exclusively with synapticvesicles within the synapse and is called v-SNARE. Together, these threeproteins form a complex that are thought to be the minimal machinery tomediate the fusion between vesicle membrane and plasma membrane. BoNT/A,E, and C¹ cleave SNAP-25, BoNT/B, D, F, G cleave synaptobrevin (Syb), atsingle but different sites. BoNT/C also cleaves syntaxin in addition toSNAP-25.

Due to their threat as a source of food poisoning, and as bioterrorismweapons, there is a need to sensitively and speedily detect BoNTs.Currently, the most sensitive method to detect toxins is to performtoxicity assay in mice. This method requires large numbers of mice, istime-consuming and cannot be used to study toxin catalytic kinetics. Anumber of amplified immunoassay systems based on using antibodiesagainst toxins have also been developed, but most of these systemsrequire complicated and expensive amplification process, and cannot beused to study toxin catalytic activity either. Although HPLC andimmunoassay can be used to detect cleaved substrate molecules andmeasure enzymatic activities of these toxins, these methods aregenerally time-consuming and complicated, some of them requirespecialized antibodies, making them inapplicable for large scalescreening. Therefore, there is a need for new and improved methods andcompositions for detecting BoNTs.

In FRET assays, two fluorogenic amino acid derivatives are used toreplace two native amino acids in a very short synthetic peptide (20-35amino acids) that contain toxin cleavage sites. The fluorescence signalof one amino acid derivative is quenched by another amino acidderivative when they are close to each other in the peptide, thismechanism is called “Förster resonance energy transfer” (FRET). Cleavageof the peptide separates the two amino acid derivatives and a decreasesin FRET can be detected.

FRET assays have been successfully used for detecting BoNTs. (See e.g.,US Pat. App. No. 2004/0191887 to Chapman, filed Oct. 28, 2003, US Pat.App. No. 2006/0134722 to Chapman, filed Dec. 20, 2004, U.S. Pat. No.7,208,285 to Steward (April 2007), and U.S. Pat. No. 7,183,066 toFernandez-Salas (February 2007), each of which is incorporated herein byreference in their entirety. Where a definition or use of a term in anincorporated reference is inconsistent or contrary to the definition ofthat term provided herein, the definition of that term provided hereinapplies and the definition of that term in the reference does notapply).

Although some success has been demonstrated in applying FRET assays todetection of BoNTs, the sensitivity and specificity are not sufficient.Improved apparatus, systems and methods are therefore needed.

SUMMARY OF THE INVENTION

The present invention provides apparatus, systems and methods in which acell is provided that includes molecular construct, where the molecularconstruct includes a donor label, an acceptor label, a linker peptidedisposed between the donor and the acceptor, the linker having acleavage site sequence, and a spacer between at least one of (a) thedonor and the cleavage site sequence and (b) the acceptor and thecleavage site sequence. The molecular construct can be cleaved by abotulinum neurotoxin, resulting in a decrease in energy transfer betweenthe donor label and the acceptor label. Comparing signals related to theenergy transfer prior to and following exposure of the cell to abotulinum neurotoxin and a candidate inhibitor provides an indication ofthe effectiveness of the candidate inhibitor.

One embodiment of the inventive concept is a method of screening for aninhibitor of a botulinum neurotoxin that includes providing a cellgenetically engineered to express a construct comprising donor label andan acceptor label positioned to provide an electronic coupling such thatthe donor can transfer energy to the acceptor by a dipole-dipolecoupling mechanism (e.g. Förster resonance energy transfer or FRET), and(a) a linker disposed between the donor label and the acceptor label,wherein the linker is a peptide sequence comprising (a) a cleavage sitepeptide comprising a SNARE motif (e.g. a SNARE mutein), (b) a firstpeptide spacer interposed between the donor and the cleavage sitepeptide, and (c) a second peptide spacer interposed between the acceptorand the cleavage site peptide, wherein the first peptide spacer and thesecond peptide spacer each comprise three to fifteen amino acids and areselected to both increase the primary structure distance between thedonor and the acceptor and reduce the tertiary structure distancebetween the donor and the acceptor relative to a corresponding constructlacking the first spacer and second spacer, such that the construct ischaracterized by increased electronic coupling between the donor and theacceptor relative to a corresponding construct lacking the first spacerand second spacer. In some embodiments the linker has a primarystructure length≥5 nm, ≥8 nm, or ≥12 nm. A first signal is characterizedfrom such a cell prior to exposure to the botulinum neurotoxin. Aftercontacting the cell with a botulinum neurotoxin in the presence of acandidate inhibitor a second signal from the cell is characterized. Thecandidate inhibitor can be considered an effective inhibitor of thebotulinum neurotoxin when comparison of the second signal to the firstsignal shows a lack of decrease in the electronic coupling.

Another embodiment of the inventive concept is a method of screening foran inhibitor of a botulinum neurotoxin that includes providing a cellgenetically engineered to express a construct comprising donor label andan acceptor label positioned to provide an electronic coupling such thatthe donor can transfer energy to the acceptor by an electronic hopbetween the donor label and the acceptor label, and (a) a linkerdisposed between the donor label and the acceptor label, wherein thelinker is a peptide sequence comprising (a) a cleavage site peptidecomprising a SNARE motif (e.g. a SNARE mutein), (b) a first peptidespacer interposed between the donor and the cleavage site peptide, and(c) a second peptide spacer interposed between the acceptor and thecleavage site peptide, wherein the first peptide spacer and the secondpeptide spacer each comprise three to fifteen amino acids and areselected to both increase the primary structure distance between thedonor and the acceptor and reduce the tertiary structure distancebetween the donor and the acceptor relative to a corresponding constructlacking the first spacer and second spacer, such that the construct ischaracterized by an increase in the electronic hop between the donor andthe acceptor relative to a corresponding construct lacking the firstspacer and second spacer. In some embodiments the linker has a primarystructure length≥5 nm, ≥8 nm, or ≥12 nm. A first signal is characterizedfrom such a cell prior to exposure to the botulinum neurotoxin. Aftercontacting the cell with a botulinum neurotoxin in the presence of acandidate inhibitor a second signal from the cell is characterized. Thecandidate inhibitor can be considered an effective inhibitor of thebotulinum neurotoxin when comparison of the second signal to the firstsignal shows a lack of decrease in the electronic coupling.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawings in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 includes two panels that provide a schematic depiction of theCFP-YFP based bio-sensors for monitoring botulinum neurotoxin proteaseactivity. Panel A of FIG. 1 depicts a design of the bio-sensorconstructs. CFP and YFP are connected via a fragment of synaptobrevin(amino acid 33-94, upper panel), or SNAP-25 (amino acid 141-206, lowerpanel), respectively. The cleavage sites for each botulinum neurotoxinon these fragments are labeled. Panel B of FIG. 1 depicts CFP and YFPfunctioning as a donor-acceptor pair for FRET, in which the excitationof CFP results in YFP fluorescence emission (upper portion of thepanel). Energy transfer between linked CFP and YFP is abolished aftercleavage of the synaptobrevin or SNAP-25 fragment with botulinumneurotoxins (lower portion of the panel). The optimal excitationwavelength for CFP is 434 nM, and the emission peak is 470 nM for CFP,and 527 nM for YFP.

FIG. 2 provides three panels that depict the fluorescence emissionspectra of the recombinant bio-sensor proteins. Panel A of FIG. 2 showsthe emission spectra of the recombinant his₆-tagged CFP and YFP alone(300 nM), as well as the mixture of these two proteins (1:1). Thefluorescence signals were collected from 450 to 550 nM using a PTIQM-1fluorometer in HEPES buffer (50 mM HEPES, 2 mM DTT, and 10 μM ZnCl₂, pH7.1). The excitation wavelength is 434 nM, the optimal for CFP. The YFPprotein only elicits a small fluorescence emission signal by directexcitation at 434 nM. Panel B of FIG. 2 shows the emission spectra ofrecombinant his₆-tagged CFP-SybII-YFP, collected as described in panelA. The arrow indicates the YFP emission peak resulted from FRET. Panel Cof FIG. 2 shows the emission spectra of recombinant his₆-taggedCFP-SNAP-25-YFP, collected as described in panel A. The arrow indicatesthe YFP emission peak resulted from FRET.

FIG. 3 provides 4 panels depicting the cleavage of bio-sensor proteinsby botulinum neurotoxin, and demonstrating that such cleavage can bemonitored by emission spectra scan in real time in vitro. Panel A ofFIG. 3 depicts results of a typical study where BoNT/B toxin waspre-reduced with 2 mM DTT, 10 μM ZnCl₂ for 30 min at 37° C. 50 nM toxinwere added to a cuvette that contained 300 nM CFP-SybII-YFP protein inthe HEPES buffer (50 mM HEPES, 2 mM DTT, 10 μM ZnCl₂). The emissionspectra was recorded as described in FIG. 2 panel A at indicated timebefore and after adding toxin (upper panel). 30 μl samples were takenfrom the cuvette after each emission scan, and mixed with SDS-loadingbuffer. These samples were subject to SDS-page and enhancedchemiluminescence (ECL). The cleavage of CFP-SybII-YFP fusion proteinwas detected using an anti-his₆ antibody that recognizes the his₆ tag atthe fusion protein N-terminus (lower portion of the panel). The cleavageof CFP-SybII-YFP fusion protein resulted in decreased YFP fluorescenceand increased CFP fluorescence. This change was recorded in real-time byemission spectra scan. Panel B of FIG. 3 depicts results of a typicalstudy where CFP-SybII-YFP was used to test BoNT/F activity, as describedin panel A. Panel C of FIG. 3 depicts results of a typical study whereCFP-SNAP-25-YFP was used to test BoNT/A activity (10 nM toxin was used),as described in panel A. Panel D of FIG. 3 depicts results of a typicalstudy where CFP-SNAP-25-YFP was used to test BoNT/E activity (10 nMtoxin was used), as described in panel A.

FIG. 4 provides three panels that depict the monitoring of botulinumneurotoxin protease kinetics using bio-sensor proteins in a microplatespectrofluorometer. Panel A of FIG. 4 shows that fluorescence changeduring the cleavage of bio-sensor proteins by botulinum neurotoxin canbe recorded in real time using a plate-reader. 10 nM BoNT/A were mixedwith 300 nM CFP-SNAP-25-YFP, and 100 μl per well sample was scannedusing a plate-reader. The excitation is 434 nm, and for each data point,both emission value at 470 nm (CFP channel), and 527 nm (YFP or FRETchannel) were collected. The reaction was traced for one and a halfhours at the interval of 30 sec per data point. The decrease of YFPfluorescence and the increase of CFP fluorescence were monitored in realtime. Panel B of FIG. 4 shows that the rate of cleavage is dependent onthe concentration of the neurotoxin. The various concentrations ofbotulinum neurotoxin A and E were tested for their ability to cleave thesame amount of bio-sensor proteins. FRET signal change (FRET ratio) ismeasured by the ratio between YFP emission signal and the CFP emissionsignal at the same data point. Panel C of FIG. 4 shows results of atypical study in which CFP-SNAP-25-YFP protein alone, and the CFP/YFPprotein mixture (1:1) were scanned at the same time, as the internalcontrol.

FIG. 5 provides two panels that demonstrate the sensitivity of thebio-sensor assay using a plate-reader. Panel A of FIG. 5 shows resultsof a typical study in which 300 nM CFP-SNAP-25-YFP were mixed withvarious concentration of BoNT/A or E in a 96-well plate, the totalvolume is 100 μl per well. The plate was incubated at 37° C. for 4 hoursand then scanned with a plate-reader (upper panel). The FRET ratio wasplotted against the log value of the toxin concentration. The EC₅₀values for each curve are listed in the table on the lower panel. Eachdata point represents the mean of three independent experiments. Panel Bof FIG. 5 shows results of a typical study in which 300 nM CFP-SybII-YFPwere mixed with various concentration of BoNT/B or F. The data werecollected and plotted as described in panel A.

FIG. 6 provides two panels that depict the monitoring of botulinumneurotoxin activity in living cells. Panel A of FIG. 6 depicts theresults of a typical study in which CFP-SNAP-25-YFP was expressed inwild type PC12 cells. The entry and catalytic activity of BoNT/A (50 nM)was monitored by recording the FRET ratio change that results fromCFP-SNAP-25-YFP cleavage inside the cells. The FRET ratio was averagedfrom a total of 53 toxin treated cells and 53 control cells. Treatmentwith BoNT/A for 72 hours reduced the FRET ratio of the entire populationof cells by a significant degree (P<1.47E-5). Panel B of FIG. 6 depictsthe results of a typical study in which PC 12 cells that express syt IIwere transfected with CFP-SybII-YFP and treated with BoNT/B (30 nM). Theentry and catalytic activity of BoNT/B were monitored by recording theFRET ratio change as in panel A; 73 toxin treated and 73 control cellswere analyzed. Treatment with BoNT/B for 72 hours reduced the FRET ratioof the entire population of cells by a significant degree (P<2E-10).

FIG. 7 provides five panels that show the monitoring BoNT/A activity inliving cells using according to the present invention. Panel A of FIG. 7depicts measuring the FRET signal of toxin sensors in living cells.CFP-SNAP-25(141-206)-YFP was used to transfect PC12 cells. This sensorappeared to be soluble in cells. Three images using different filter set(CFP, FRET and YFP) were taken for each cell sequentially, using exactlythe same settings. Images were color coded to reflect the fluorescenceintensity in arbitrary units as indicated in the look-up table on theleft. The corrected FRET value was calculated by subtracting thecross-talk from both CFP and YFP from the signals collected using theFRET filter set, as detailed in the Methods. Panel B of FIG. 7 depictsresults of a typical study in which PC12 cells transfected withCFP-SNAP-25(141-206)-YFP were used to detect BoNT/A activity. Fifty nMBoNT/A holotoxin was added to the culture medium and 80 cells wereanalyzed after 96 hours. The corrected FRET signal was normalized to theCFP fluorescence signal and plotted as a histogram with the indicatedbins. Control cells were transfected with the same sensor but were nottreated with toxins, and they were analyzed in parallel. Incubation withBoNT/A shifted the FRET ratio (corrected FRET/CFP) among the cellpopulation, indicating the sensor proteins were cleaved by BoNT/A incells. However, the shift was small, indicating that the cleavage wasnot efficient in cells. Panel C of FIG. 7 depicts results of a study inwhich an efficient toxin sensor was built by linking CFP and YFP throughfull-length SNAP-25 (amino acid 1-206, left side of panel), and testedfor detecting BoNT/A activity in cells. This CFP-SNAP-25(FL)-YFP fusionprotein was localized primarily to plasma membranes in cells viapalmitoylation at its four cysteines (left side and middle of panel).Photomicrographs of the middle portion of the panel show PC12 cells thatwere transfected with the CFP-SNAP-25(FL)-YFP sensor and used to detectBoNT/A activity. Fifty nM BoNT/A holotoxin was added to the culturemedium and the FRET signals of 200 cells were analyzed after 48 and 96hours as described in panel A. Control cells were transfected with toxinsensors but were not treated with toxins, and they were analyzed inparallel. Images of representative cells are shown. This sensor yieldedsignificant FRET (upper “corrected FRET” micrograph of the middleportion of the panel). The FRET signal was abolished after cells weretreated with BoNT/A (96 h, lower “corrected FRET” micrograph of themiddle portion of the panel). One of the cleavage products, theC-terminus of SNAP-25 tagged with YFP, was degraded after toxincleavage. Thus, the fluorescence signal of YFP was significantlydecreased in toxin-treated cells (lower “YFP channel” micrograph of themiddle portion of the panel). FRET ratios are plotted as a histogramwith indicated bins as described in panel B in the right portion of thepanel. Panel D of FIG. 6 depicts the results of a typical study in whichPC12 cells were transfected with CFP-SNAP-25(Cys-Ala)-YFP (full lengthSNAP-25 with Cys 85, 88, 90, 92 Ala mutations, left side). This proteinhas diffusely distributed throughout the cytosol, and lacked the strongFRET signal observed for CFP-SNAP-25(FL)-YFP (right side, “correctedFRET” micrograph). Panel E of FIG. 6 depicts the results of a typicalstudy in which PC12 cells were transfected with CFP-SNAP-25(FL)-YFP andCFP-SNAP-25(Cys-Ala)-YFP. Cells were then treated with (+, intact cells)or without (−, intact cells) BoNT/A (50 nM, 72 h), and were harvested.Half of the cell extracts from samples that are not been exposed toBoNT/A were also incubated with (+, in vitro) or without (−, in vitro)reduced BoNT/A in vitro (200 nM, 30 min, 37° C.), served as controls toshow the cleavage products (two cleavage products are indicated byarrows). The same amount of each sample (30 μg cell lysate) was loadedto one SDS-page gel and subjected to immunoblot analysis using ananti-GFP antibody. While CFP-SNAP-25(FL)-YFP underwent significantcleavage in intact cells, there was no detectable cleavage ofCFP-SNAP-25(Cys-Ala)-YFP in cells, indicating the membrane anchoring isimportant for efficient cleavage by BoNT/A in living cells. Only onecleavage product (CFP-SNAP-25(1-197)) was detected in toxin treatedcells, indicating that the other cleavage product (SNAP-25(198-206)-YFP)was largely degraded in cells.

FIG. 8 provides three panels that show anchoringCFP-SNAP-25(141-206)-YFP sensor to the plasma membrane created a sensorthat was efficiently cleaved by BoNT/A in cells. Panel A of FIG. 8provides a schematic description of the construct built to targetCFP-SNAP-25(141-206)-YFP to the plasma membrane. A fragment of SNAP-25that contains the palmitoylation sites (residues 83-120) was fused tothe N-terminus of the CFP-SNAP-25(141-206)-YFP sensor, and this fragmenttargeted the fusion protein to the plasma membrane. Panel B of FIG. 8shows results of a typical study in which PC12 cells were transfectedwith SNAP-25(83-120)-CFP-SNAP-25(141-206)-YFP. Fifty nM BoNT/A holotoxinwas added to the culture medium and the FRET signals of 80 cells wereanalyzed after 96 hours as described in panel A of FIG. 7. Controlcells, transfected with toxin sensors but not treated with toxins, wereanalyzed in parallel. The images of representative cells are shown inthe left panel. This sensor yielded significant FRET (upper “correctedFRET” frame of the left panel). The FRET signal was reduced after cellswere treated with BoNT/A (96 h, lower “corrected FRET” frame of the leftpanel). Right panel: the FRET ratios of cells are plotted as a histogramwith indicated bins as described in panel B of FIG. 7. Panel C of FIG. 8shows results of a typical study in which PC12 cells were transfectedwith various CFP/YFP constructs and the corresponding FRET ratios weredetermined as described in panel A of FIG. 7. Co-expression of CFP andYFP in cells, did not result in significant FRET under our assayconditions. CFP-SNAP-25(FL)-YFP exhibited significant levels of FRETwhereas the soluble CFP-SNAP-25(Cys-Ala)-YFP did not.

FIG. 9 provides four panels that show efficient cleavage of Syb byBoNT/B requires the localization of Syb to vesicles. Panel A of FIG. 9shows results of a typical study in which CFP-Syb(33-94)-YFP was used totransfect a PC12 cell line that stably expresses synaptotagmin II (Donget al. Synaptotagmins I and II mediate entry of botulinum neurotoxin Binto cells. J. Cell Biol. 162, 1293-1303 (2003)). This sensor appears tobe soluble inside cells and generates strong FRET signals (upper panel).PC12 cells transfected with CFP-Syb(33-94)-YFP were used to detectBoNT/B activity. Fifty nM BoNT/B holotoxin was added to the culturemedium and 80 cells were analyzed after 96 hours as described in panel Bof FIG. 7. Control cells were transfected with the same sensor but werenot treated with toxins, and they were analyzed in parallel. Incubationwith BoNT/B shifted the FRET ratio among the cell population, indicatingthe sensor proteins were cleaved by BoNT/B in cells. However, the shiftwas small, indicating that the cleavage was not efficient in cells.Panel B of FIG. 9 provides a schematic description of YFP-Syb(FL)-CFPsensor. Full-length Syb contain 116 amino acids, and is localized tovesicles through a single transmembrane domain. Cleavage of Syb byBoNT/B released the cytoplasmic domain of Syb tagged with YFP from thevesicle. Panel C of FIG. 9 shows results of a typical study in whichPC12 cells that stably express synaptotagmin II were transfected withYFP-Syb(FL)-CFP, and were treated with BoNT/B (50 nM, 48 h, lowerframes), or without toxin (control, upper frames). CFP and YFPfluorescence images were collected for each cell, and representativecells are shown. This sensor is localized to vesicles, and was excludedfrom the nucleus in living cells, as evidenced by both CFP and YFPfluorescent signals (upper frames). BoNT/B treatment resulted in aredistribution of YFP signals, which became soluble in the cytosol andentered the nucleus. Panel D of FIG. 9 shows results of a typical studyin which a truncated version of Syb, residues 33-116, was used to link aCFP and YFP. This construct contains the same cytosolic region (residues33-94, panel (b)) as the Syb fragments in the soluble sensorCFP-Syb(33-96)-YFP, and it also contains the transmembrane domain ofSyb. PC12 cells that express synaptotagmin II were transfected withCFP-Syb(33-116)-YFP and CFP-Syb(33-94)-YFP. Cells were then treated with(+, intact cells) or without (−, intact cells) BoNT/B (50 nM, 48 h), andwere harvested. Half of the cell extracts from samples that were notexposed to BoNT/B were also incubated with (+, in vitro) or without (−,in vitro) reduced BoNT/B in vitro (200 nM, 30 min, 37° C.). Two cleavageproducts are indicated by asterisks. The same amount of each sample (30μg cell lysate) was loaded to one SDS-page gel and subjected toimmunoblot analysis using an anti-GFP antibody. WhileCFP-Syb(33-116)-YFP underwent significant cleavage in intact cells,there was no detectable cleavage of CFP-Syb(33-94)-YFP, indicating thelocalization to vesicles is important for efficient cleavage by BoNT/Bin living cells.

FIG. 10 is a schematic depicting a prior art construct, having a linkerthat includes a cleavage site sequence disposed between a donor labeland an acceptor label.

FIG. 11A is a schematic depicting one embodiment of the construct of thepresent invention, having a linker that includes a cleavage sitesequence and a spacer, the spacer being disposed between the cleavagesite sequence and the acceptor label.

FIG. 11B is a schematic depicting an alternative embodiment of theconstruct of the present invention, having a linker that includes acleavage site sequence and a spacer, the spacer being disposed betweenthe donor label and the cleavage site sequence.

FIG. 11C is a schematic depicting another alternative embodiment of theconstruct of the present invention, having a linker that includes acleavage site sequence and a spacer, the spacer being disposed betweenthe donor and the cleavage site sequence and cleavage site sequence andthe acceptor label.

FIG. 12 is a block diagram illustrating the steps of a method ofimproving sensitivity of energy transfer between a donor label and anacceptor label using the construct of the present invention.

FIG. 13 is a block diagram illustrating the steps of a method fordetecting botulinum neurotoxin using the construct of the presentinvention.

DETAILED DESCRIPTION

The present invention provides novel compositions and methods based onfluorescence resonance energy transfer (FRET) between fluorophoreslinked by a peptide linker which is a substrate of a BoNT and can becleaved by the toxin, to detect botulinum neurotoxins and monitor theirsubstrate cleavage activity, preferably in real time. The method andcompositions of the present invention allow for the detection ofpico-molar level BoNTs within hours, and can trace toxin enzymatickinetics in real time. The methods and compositions can further be usedin high-throughput assay systems for large-scale screening of toxininhibitors, including inhibitors of toxin cellular entry andtranslocation through vesicle membrane using cultured cells. The presentinvention is also suitable for monitoring botulinum neurotoxin activityin living cells and neurons.

In another embodiment, the present invention provides a construct andmethod of using the construct which comprises full-length SNAP-25 andSyb proteins as the linkers, as fluorescent biosensors that can detecttoxin activity within living cells. Cleavage of SNAP-25 abolishedCFP/YFP FRET signals and cleavage of Syb resulted in spatialredistribution of the YFP fluorescence in cells. The present inventionprovides a means to carry out cell based screening of toxin inhibitorsand for characterizing toxin activity inside cells. The presentinvention also discloses that the sub-cellular localization of SNAP-25and Syb affects efficient cleavage by BoNT/A and B in cells,respectively.

Fluorescent Resonance Energy Transfer (FRET) is a tool which allows theassessment of the distance between one molecule and another (e.g. aprotein or nucleic acid) or between two positions on the same molecule.FRET is now widely known in the art (for a review, see Matyus, (1992) J.Photochem. Photobiol. B: Biol., 12:323). FRET is a radiationless processin which energy is transferred from an excited donor molecule to anacceptor molecule. Radiationless energy transfer is thequantum-mechanical process by which the energy of the excited state ofone fluorophore is transferred without actual photon emission to asecond fluorophore. The quantum physical principles are reviewed inJovin and Jovin, 1989, Cell Structure and Function byMicrospectrofluorometry, eds. E. Kohen and J. G. Hirschberg, AcademicPress. Briefly, a fluorophore absorbs light energy at a characteristicwavelength. This wavelength is also known as the excitation wavelength.The energy absorbed by a fluorochrome is subsequently released throughvarious pathways, one being emission of photons to produce fluorescence.The wavelength of light being emitted is known as the emissionwavelength and is an inherent characteristic of a particularfluorophore. In FRET, that energy is released at the emission wavelengthof the second fluorophore. The first fluorophore is generally termed thedonor (D) and has an excited state of higher energy than that of thesecond fluorophore, termed the acceptor (A).

An essential feature of the process is that the emission spectrum of thedonor overlap with the excitation spectrum of the acceptor, and that thedonor and acceptor be sufficiently close.

In addition, the distance between D and A must be sufficiently small toallow the radiationless transfer of energy between the fluorophores.Because the rate of energy transfer is inversely proportional to thesixth power of the distance between the donor and acceptor, the energytransfer efficiency is extremely sensitive to distance changes. Energytransfer is said to occur with detectable efficiency in the 1-10 nmdistance range, but is typically 4-6 nm for optimal results. Thedistance range over which radiationless energy transfer is effectivedepends on many other factors as well, including the fluorescencequantum efficiency of the donor, the extinction coefficient of theacceptor, the degree of overlap of their respective spectra, therefractive index of the medium, and the relative orientation of thetransition moments of the two fluorophores.

The present invention provides a construct (“FRET construct”) whichcomprises a fluorophore FRET donor and an acceptor linked by linkerpeptide (“substrate peptide”) that is cleavable by a corresponding BoNT.In the presence of a BoNT, the linker peptide is cleaved, therebyleading to a decrease in energy transfer and increased emission of lightby the donor fluorophore. In this way, the proteolysis activity of thetoxin can be monitored and quantitated in real-time.

As used herein with respect to donor and corresponding acceptorfluorescent moieties, “corresponding” refers to an acceptor fluorescentmoiety having an emission spectrum that overlaps the excitation spectrumof the donor fluorescent moiety. The wavelength maximum of the emissionspectrum of the acceptor fluorescent moiety should be at least 100 nmgreater than the wavelength maximum of the excitation spectrum of thedonor fluorescent moiety. Accordingly, efficient non-radioactive energytransfer can be produced.

As used herein with respect to substrate peptide and BoNT,“corresponding” refers to a BoNT toxin that is capable of acting on thelinker peptide and cleaves at a specific cleavage site.

Fluorescent donor and corresponding acceptor moieties are generallychosen for (a) high efficiency Forster energy transfer; (b) a largefinal Stokes shift (>100 nm); (c) shift of the emission as far aspossible into the red portion of the visible spectrum (>600 nm); and (d)shift of the emission to a higher wavelength than the Raman waterfluorescent emission produced by excitation at the donor excitationwavelength. For example, a donor fluorescent moiety can be chosen thathas its excitation maximum near a laser line (for example,Helium-Cadmium 442 nm or Argon 488 nm), a high extinction coefficient, ahigh quantum yield, and a good overlap of its fluorescent emission withthe excitation spectrum of the corresponding acceptor fluorescentmoiety. A corresponding acceptor fluorescent moiety can be chosen thathas a high extinction coefficient, a high quantum yield, a good overlapof its excitation with the emission of the donor fluorescent moiety, andemission in the red part of the visible spectrum (>600 nm).

A skilled artisan will recognize that many fluorophore molecules aresuitable for FRET. In a preferred embodiment, fluorescent proteins areused as fluorophores. Representative donor fluorescent moieties that canbe used with various acceptor fluorescent moieties in FRET technologyinclude fluorescein, Lucifer Yellow, B-phycoerythrin,9-acridineisothiocyanate, Lucifer Yellow VS,4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid,7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimdyl1-pyrenebutyrate, and4-acetamido-4′-isothiocyanatostilbene-2-,2′-disulfonic acid derivatives.Representative acceptor fluorescent moieties, depending upon the donorfluorescent moiety used, include LC-Red 640, LC-Red 705, Cy5, Cy5.5,Lissamine rhodamine B sulfonyl chloride, tetramethyl rhodamineisothiocyanate, rhodamine×isothiocyanate, erythrosine isothiocyanate,fluorescein, diethylenetriamine pentaacetate or other chelates ofLanthanide ions (e.g., Europium, or Terbium). Donor and acceptorfluorescent moieties can be obtained, for example, from Molecular Probes(Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

Table 1 lists other examples of chemical fluorophores suitable for usein the invention, along with their excitation and emission wavelengths.

Certain naturally occurring amino acids, such as tryptophan, arefluorescent. Amino acids may also be derivatized, e.g. by linking afluorescent group onto an amino acid (such as linking AEDANS to a Cys),to create a fluorophore pair for FRET. The AEDANS-Cys pair is commonlyused to detect protein conformational change and interactions. Someother forms fluorescence groups have also been used to modify aminoacids and to generate FRET within the protein fragments (e.g.2,4-dinitrophenyl-lysine withS—(N-[4-methyl-7-dimethylamino-coumarin-3-yl]-carboxamidomethyl)-cysteine-).

In another embodiment, which is especially suitable for using in livecells, green fluorescent protein (GFP) and its various mutants are usedas the fluorophores. Examples of fluorescent proteins which vary amongthemselves in excitation and emission maxima are listed in Table 1 of WO97/28261 (Tsien et al., 1997), which is incorporated herein byreference. These (each followed by [excitation max./emission max.]wavelengths expressed in nanometers) include wild-type Green FluorescentProtein [395(475)/508] and the cloned mutant of Green FluorescentProtein variants P4 [383/447], P4-3 [381/445], W7 [433(453)/475(501)],W2 [432(453)/408], S65T [489/511], P4-1 [504(396)/480], S65A [471/504],S65C [479/507], S65L [484/510], Y66F [360/442], Y66W [458/480], IOc[513/527], WIB [432(453)/476(503)], Emerald [487/508] and Sapphire[395/511]. Red fluorescent proteins such as DsRed (Clontech) having anexcitation maximum of 558 nm and an emission maximum of 583 can also beused. This list is not exhaustive of fluorescent proteins known in theart; additional examples are found in the Genbank and SwissPro publicdatabases.

TABLE 1 Fluorophore Excitation (nm) Emission (nm) Color PKH2 ™ 490 504green PKH67 ™ 490 502 green Fluorescein (FITC) 495 525 green HOESCHT33258 ™ 360 470 blue R-Phycoerythrin (PE) 488 578 orange-red RHODAMINE(TRITC) 552 570 red QUANTUMRED ™ 488 670 red PKH26 ™ 551 567 redTEXASRED ™ 596 620 red CY3 ™ 552 570 red

GFP is a 27-30 KD protein, and can be fused with another protein, e.g.the target protein, and the fusion protein can be engineered to beexpressed in a host cell, such as those of E. coli. GFP and its variousmutants are capable of generating fluorescence in live cells andtissues. The mutation forms of GFP has slight amino acid differenceswithin their fluorescence region which result in shifted spectrum. Moremutations of GFP are expected to be created in the future to havedistinct spectra. Among these GFP variants, BFP-YFP, BFP-CFP, CFP-YFP,GFP-DsRed, are commonly used as FRET donor-acceptor pairs to detectprotein-protein interactions. These pairs are also suitable to detectprotease cleavage of the linker region that links the pair.

The use of fluorescent proteins is preferred because they enable the useof a linker fragment of about 60 amino acids residues. Longer fragmentsusually are more sensitive to toxin recognition and cleavage, thus,results in a higher sensitivity for detecting toxin. As shown in theexamples below, the EC50 for BoNT/A and E after 4 hour incubation withCFP-SNAP-YFP are as low as 15-20 pM (1-2 ng/ml), when measured with awidely used microplate spectrofluorometer (SPECTRAMAX GEMINI™, MolecularDevices). EC50 for BoNT/B and F are about 200-250 pM, and thesensitivity can be enhanced by increasing incubation time.

According to one embodiment of the present invention, two fluorophoresare linked together by a linker of suitable length, such that FREToccurs. The linker is a fragment of a BoNT substrate protein. Whenexposed to a BoNT capable of cleaving the linker fragment, the twofluorophores are separated and FRET is abolished. The present inventionprovides accordingly a method for detecting BoNT by detecting the changein FRET. SNARE proteins from many species are suitable as substrateproteins for BoNT toxins, because these proteins are known to beconserved at the amino acid level. Many of these BoNT substrate proteinsare known and available to be used or modified for use as a suitablelinker peptide for the present invention. Some of the substrate proteinsand their GenBank accession numbers are listed in

TABLE 1 Protein Origin Accession # syb 1 mouse NP-033522 svb 1a humanNP_055046 syb 1 rat AAN85832 syb African frog AAB88137 syb electric rayA32146 syb California sea hare P35589 syb Takifugu rubripes AAB94047 sybdrosophila AAB28707 syb II mouse NP_033523 syb II African frog P47193Syb II rabbit AAN14408 syb II rat NP-036795 syb II human AAH19608 syb 3human AAP36821 SNAP25-1 Zebra fish AAC64289 SNAP25-A human NP-003072SNAP25a American frog AAO13788 SNAP25 mouse XP_130450 SNAP25 ratNP_112253 SNAP25 goldfish 150480 SNAP25-b Zebra fish NP_571509 SNAP25bAmerican frog AAO13789 SNAP25-3 human CAC34535

Each BoNT toxin is known to cleave a specific peptide bond between twospecific amino acids within the toxin cleavage site. Table 3 below liststhe amino acid pairs for each BoNT toxin. These pairs of amino acidsequence, however, are not sufficient for toxin recognition andcleavage. For example, BoNT/A cleaves SNAP-25 at Q(197)-R(198) of therat SNAP-25 sequence (GenBank accession No: NP-112253), but notQ(15)-R(16). Generally, there is no conserved amino acid sequence as therecognition site; rather, the toxins are believed to recognize thetertiary, rather than the primary, structure of their target protein.Nevertheless, a very short fragment of the substrate protein issufficient for toxin recognition and cleavage, regardless of its speciesorigin, as long as they have the two amino acid residues at the toxincleavage site listed above in Table 3 below.

The linker protein or peptide can be as long as the full-length of theBoNT substrate protein. Preferably the linker is a shorter fragment ofthe substrate protein. A full-length substrate linker may be too longfor efficient FRET, and a shorter fragment is more effective and easierto produce than the full-length protein. On the other hand, as indicatedabove, the linker peptide should be above certain minimum length,because below such a minimum length, cleavage of the linker peptide bythe respective BoNTs becomes inefficient.

TABLE 3  Peptide Bonds Recognized and Cleaved by BoNT Toxins CleavagePutative Minimum Toxin Site Recognition Sequence BoNT/A Q-RGlu-Ala-Asn-Gln- (SEQ ID  Arg-Ala-Thr-Lys NO: 1) BoNT/B Q-FGly-Ala-Ser-Gln-Phe- (SEQ ID  Glu-Thr-Ser NO: 2) BoNT/C R-AAla-Asn-Gln-Arg-Ala- (SEQ ID  (SNAP25) Thr-Lys-Met NO: 3) BoNT/C K-AAsp-Thr-Lys-Lys-Ala- (SEQ ID  (Syntaxin) Val-Lys-Phe NO: 4) BoNT/D K-LBoNT/E R-I Gln-Ile-Asp-Arg-Ile- (SEQ ID  Met-Glu-Lys NO: 5) BoNT/F Q-KGlu-Arg-Asp-Gln- (SEQ ID  Lys-Leu-Ser-Glu NO: 6) BoNT/G A-A

Using syb II and BoNT/B as an example, Table 4 below illustrates therelationship between linker-peptide length and toxin cleavage rate. Thefull-length rat syb II protein (GenBank No: NP-036795) has 116 aminoacids, of which amino acid 1-94 at the amino terminus is the cytoplasmicdomain and the rest is the transmembrane domain. As Table 1 makes clear,within certain limit, a shorter fragment is cleaved by the toxin at aslower rate (data from Foran et al., Biochemistry 33:15365, 1994).

As can be seen from Table 4, tetanus neurotoxin (TeNT) requires a longerfragment (33-94) for optimum cleavage than BoNT/B (55-94). A fragmentconsisting of 60-94 has been used in several studies including severalpeptide-based toxin assay methods (Schmidt et al., 2003, supra, andSchmidt et al., 2001, Analytical Biochemistry, 296: 130-137).

For BoNT/A, the 141-206 fragment of SNAP-25 is required for retainingmost of the toxin sensitivity (Washbourne et al., 1997, FEBS Letters,418:1). There are also other reports that a shorter peptide, amino acids187-203 of SNAP25, is sufficient to be cleaved by BoNT/A (2001). Theminimum site for BoNT/A is: Glu-Ala-Asn-Gln-Arg-Ala-Thr-Lys (SEQ ID NO:1). BoNT/A cleave Gln-Arg.

TABLE 4 Relationship Between Syb II fragment Length and Cleavage RateSyb II Fragment Relative cleavage rate by % Relative cleavage rate byLength BoNT/B TeNT full length 1-116 100 (%) 100 (%) 33-94 100 100 45-94121 1.1 55-94 105 0.4 65-94 7 0.3

Using full-length SNAP-25 as the linker sequence between CFP and YFPinside PC12 cells, preliminary results indicate that FRET signalsobtained are stronger than those obtained using a shorter fragment,enough to be detected using a conventional lab microscope. It isbelieved that in PC12 cells the rate of cleavage of full-length SNAP-25by BoNT/A is faster and more consistent from cell to cell than the shortfragment, likely due to the fact that full-length SNAP-25 is targetedonto plasma membrane, on to which the BoNT/A light chain may also betargeted and anchored.

For BoNT/B, a fragment as short as between residues 60-94 was found tobe as effective as a fragment between residues 33-94. Preferably, afragment between 33-94 is used for BoNT/B and TeNT. Both toxins cleavebetween Gln and Phe, and the minimum sequence for cleavage is believedto be: Gly-Ala-Ser-Gln-Phe-Glu-Thr-Ser (SEQ ID NO: 2). There areindications that BoNT/B light chain may be targeted and anchored onsynaptic vesicles, it may be desirable to also target, via signalsequences, a FRET construct of the present invention onto synapticvesicles to achieve increased cleavage efficient inside cells.

BoNT/C cleaves both SNAP25 and Syntaxin, and is believed to cleave at avery slow rate if the substrate is in solution. Native SNAP25 andSyntaxin that reside on the cell membrane are cleaved most efficientlyby BoNT/C. The minimum cleavage sequence for SNAP25 is:Ala-Asn-Gln-Arg-Ala-Thr-Lys-Met (SEQ ID NO: 3), where cleavage occursbetween Arg-Ala; for Syntaxin, the minimum cleavage sequence isAsp-Thr-Lys-Lys-Ala-Val-Lys-Phe (SEQ ID NO: 4), and cleavage occurs atLys-Ala.

BoNT/E requires a minimum sequence of: Gln-Ile-Asp-Arg-Ile-Met-Glu-Lys(SEQ ID NO: 5), and cleaves between Arg-Ile.

BoNT/F cleaves Gln-Lys. Schmidt et al. (Analytical Biochemistry, 296:130-137 (2001)) reported that a 37-75 fragment of syb II retains most oftoxin sensitivity, and the minimum sequence is:Glu-Arg-Asp-Gln-Lys-Leu-Ser-Glu (SEQ ID NO: 6).

From the above discussion on the minimum cleavage sites and therelationship between FRET signal strength and linker length, and betweencleavage efficiency and linker length, a person skilled in the art caneasily choose suitable linker length to achieve optimal balance betweenFRET signal strength and cleavage efficiency.

Preferably, the linker length is anywhere between about 8 a.a. to about100 a.a., preferably between 10-90, more preferable between 20-80,between 30-70, between 40-60 a.a. long, depending on the specificsubstrate and toxin combination.

In one embodiment, a linker protein or fragments thereof may be firstpurified, or peptides were first synthesized, and then the fluorescencegroups were added onto certain amino acids through chemical reaction. Afluorescent label is either attached to the linker polypeptide or,alternatively, a fluorescent protein is fused in-frame with a linkerpolypeptide, as described below. The above discussion makes clear thatwhile short substrate fragments are desirable for toxin detectionspecificity, longer fragments may be desirable for improved signalstrength or cleavage efficiency. It is readily recognized that when thesubstrate protein contains more than one recognition site for one BoNT,a position result alone will not be sufficient to identify whichspecific toxin is present in the sample. In one embodiment of thepresent invention, if a longer substrate fragment, especially afull-length substrate protein, is used, the substrate may be engineered,e.g. via site-directed mutagenesis or other molecular engineeringmethods well-known to those skilled in the art, such that it containsonly one toxin/protease recognition site. See e.g. Zhang et al, 2002,Neuron 34:599-611 “Ca2+-dependent synaptotagmin binding to SNAP-25 isessential for Ca2+ triggered exocytosis” (showing that SNAP-25 havingmutations at BoNT/E cleavage site (Asp 179 to Lys) is resistant toBoNT/E cleavage, but behaves normally when tested for SNARE complexformation). In a preferred embodiment, the method of the presentinvention uses a combination of specificity engineering and lengthoptimization to achieve optimal signal strength, cleavage efficiency andtoxin/serotype specificity.

In a preferred embodiment, the fluorophores are suitable fluorescentproteins linked by a suitable substrate peptide. A FRET construct maythen be produced via the expression of recombinant nucleic acidmolecules comprising an in-frame fusion of sequences encoding such apolypeptide and a fluorescent protein label either in vitro (e.g., usinga cell-free transcription/translation system, or instead using culturedcells transformed or transfected using methods well known in the art).Suitable cells for producing the FRET construct may be a bacterial,fungal, plant, or an animal cell. The FRET construct may also beproduced in vivo, for example in a transgenic plant, or in a transgenicanimal including, but not limited to, insects, amphibians, and mammals.A recombinant nucleic acid molecule of use in the invention may beconstructed and expressed by molecular methods well known in the art,and may additionally comprise sequences including, but not limited to,those which encode a tag (e.g., a histidine tag) to enable easypurification, a linker, a secretion signal, a nuclear localizationsignal or other primary sequence signal capable of targeting theconstruct to a particular cellular location, if it is so desired.

As low as 300 nM proteins is enough to generate sufficient fluorescencesignals that can be detected using a microplate spectrofluorometer. Thefluorescence signal change can be traced in real time to reflect thetoxin protease enzymatic activity. Real time monitoring measures signalchanges as a reaction progresses, and allows both rapid data collectionand yields information regarding reaction kinetics under variousconditions. FRET ratio changes and degrees of cleavage may becorrelated, for example for a certain spectrofluorometer using a methodsuch as HPLC assay in order to correlate the unit of kinetic constantfrom the FRET ratio to substrate concentration.

FIG. 10, is a schematic of a prior art construct 100, having a linker130 that includes a cleavage site sequence 140 and cleavage site 142disposed between a donor label 110 and an acceptor label 120. This typeof prior art construct works reasonably well for detecting BoNTs.However, exemplary construct 100 a of FIG. 11a , surprisingly, has anenhanced sensitivity for detecting BoNTs due the inclusion of spacer 150a within linker 130 a.

FIG. 11a is a schematic depicting one embodiment of the construct 100 aof the present invention, having a linker 130 a that includes a cleavagesite sequence 140, a cleavage site 142, and a spacer 150 a. Spacer 150 ais disposed between the cleavage site sequence 140 and the acceptorlabel 120. Preferably, construct 100 a is selected from the groupconsisting of CFP-(SGLRSRA)(SEQ ID NO. 9)-SNAP-25-(SNS)-YFP, andCFP-(SGLRSRA)(SEQ ID NO. 9)-synaptobrevin-(SNS)-YFP.

Donor label 110 and acceptor label 120 are positioned to provide anelectronic coupling such that the donor label can transfer energy to theacceptor label by a dipole-dipole coupling mechanism, including but notlimited to Förster resonance energy transfer (FRET).

Linker peptide 130 a is a substrate of a botulinum neurotoxin selectedfrom the group consisting of synaptobrevin (VAMP), syntaxin and SNAP-25,or a fragment thereof that can be recognized and cleaved by thebotulinum neurotoxin. These proteins collectively are referred to as theSNARE proteins. Linker 130 a can have a primary structure length of anysuitable length, including for example, greater than or equal to 5 nm, 8nm, 10 nm, 12 nm, 14 nm, and 20 nm.

Spacer 150 a can have any suitable number of amino acids, but preferablyat least 3, 5, 7, 10, 12, or 15 amino acids. Spacer 150 a can include asequence selected from the group consisting of (GGGGS)n (SEQ ID NO. 7)and (EAAAK)n (SEQ ID NO. 8), where n is 1-3. Alternatively, spacer 150 acan comprise a SNARE protein, motif, or mutein. Although spacer 150 aincreases the primary structure distance between donor label 110 andacceptor label 120, spacer 150 a advantageously increases the electroniccoupling (FRET effect) between donor label 110 and acceptor label 120relative to a corresponding construct without the spacer. The enhancedelectronic coupling occurs because spacer 150 a reduces the tertiarystructure distance between donor label 110 and acceptor label 120, thusallowing increase electronic coupling.

Cleavage site sequence 140 can comprise (a) a SNARE protein, motif,mutein, and (b) a spacer with at least 5 amino acids, wherein the spacerincludes a sequence selected from the group consisting of (GGGGS)n (SEQID NO. 7) and (EAAAK)n (SEQ ID NO. 8), where n is 1-3.

Construct 100 b of FIG. 11b is similar to construct 100 a of FIG. 11aexcept that linker 130 b has spacer 150 b disposed between the donorlabel 110 and the cleavage site sequence 140.

Construct 100 c of FIG. 11e is similar to construct 100 a of FIG. 11aexcept that linker 130 b has (a) spacer 150 c disposed between donorlabel 110 and cleavage site sequence 140 and (b) spacer 150 d disposedbetween cleavage site sequence 140 and acceptor label 120.

FIG. 12 illustrates the steps of a method 200 for improving thesensitivity of energy transfer between the donor label and the acceptorlabel using the construct of FIGS. 11a-11c . Step 210 is comprised ofproviding a construct according to FIGS. 11a-11c comprising the donorlabel and the acceptor label being physically coupled through a linker.The construct is a protein based construct, and the linker is a peptidesequence 212. Step 220 is comprised of including in the linker acleavage site sequence. Optionally the cleavage site sequence comprisesa SNARE protein, or a fragment or mutein thereof, and the spacercomprises at least five amino acids 222. Step 230 is comprised ofincluding a spacer in the linker between at least one of the donor andthe cleavage site sequence and the acceptor and the cleavage sitesequence, whereby electronic coupling between the donor and the acceptoris increased by (a) reducing a tertiary structure distance between thedonor and the acceptor 234, and (b) providing an electronic hop betweenthe donor and the acceptor 236. Optionally, the spacer includes asequence selected from the group consisting of (GGGGS)n (SEQ ID NO. 7)and (EAAAK)n (SEQ ID NO. 8), where n is 1-3 232.

FIG. 13 illustrates the steps of a method 300 for detecting botulinumneurotoxin using the construct of FIGS. 11a-11c . Step 310 is comprisedof providing a construct of claim 1, wherein a linker is a substrateprotein or a cleavable fragment thereof of the botulinum neurotoxin tobe detected. Step 320 is comprised of exposing the construct to a samplesuspected of containing the botulinum neurotoxin under a condition underwhich the botulinum neurotoxin cleaves the substrate protein or thefragment thereof. And step 330 is comprised of detecting and comparing aFRET signal before and after the construct is exposed to the sample,wherein a decrease in FRET indicates the presence of botulinumneurotoxin in the sample.

The method of the present invention is highly sensitive, and as aconsequence, can be used to detect trace amount of BoNTs inenvironmental samples directly, including protoxins inside Botulinumbacterial cells. Accordingly, the present invention further provides amethod for toxin detection and identification directly usingenvironmental samples.

The present invention further provides a method for screening forinhibitors of BoNTs using the above described in vitro system. Becauseof its high sensitivity, rapid readout, and ease of use An in vitrosystems based on the present invention is also suitable for screeningtoxin inhibitors. Specifically, a suitable BoNT substrate-FRET constructis exposed to a corresponding BoNT, in the presence of a candidateinhibitor substance, and changes in FRET signals are monitored todetermine whether the candidate inhibits the activities of the BoNT.

The present invention further provides for a method for detecting a BoNTusing a cell-based system for detecting BoNTs and further for screeningfor inhibitors of BoNTs. A suitable BoNT substrate-FRET construct asdescribed above is expressed inside a cell, and the cell is then exposedto a sample suspected of containing a BoNT, and changes in FRET signalsare then monitored as an indication of the presence/absence orconcentration of the BoNT. Specifically, a decrease in FRET signalsindicates that the sample contains a corresponding BoNT.

Cell-based high-throughput screening assays have the potential to revealnot only agents that can block proteolytic activity of the toxins, butalso agents that can block other steps in the action of the toxin suchas binding to its cellular receptor(s), light chain translocation acrossendosomal membranes and light chain refolding in the cytosol aftertranslocation.

The present invention further provides a method for screening forinhibitors of BoNTs using the above described cell-based system.Specifically, a cell expressing a suitable BoNT substrate-FRET constructis exposed to a corresponding BoNT, in the presence of a candidateinhibitor substance, and changes in FRET signals are monitored todetermine whether the candidate inhibits the activities of the BoNT.Compared to other in vitro based screening methods which can onlyidentify direct inhibitors of toxin-substrate interaction, thecell-based screening method of the present invention further allows forthe screening for inhibitors of other toxin-related activities, such asbut not limited to toxin-membrane receptor binding, membranetranslocation, and intra cellular toxin movement.

According to a preferred embodiment, a recombinant nucleic acidmolecule, preferably an expression vector, encoding a BoNT substratepolypeptide and two suitable FRET-effecting fluorescent peptides isintroduced into a suitable host cell. An ordinarily skilled person canchoose a suitable expression vector, preferably a mammalian expressionvector for the invention, and will recognize that there are enormousnumbers of choices. For example, the pcDNA series of vectors, such aspCI and pSi (from Promega, Madison, Wis.), CDM8, pCeo4. Many of thesevectors use viral promoters. Preferably, inducible promoters are used,such as the tet-off and tet-on vectors from BD Biosciences (San Jose,Calif.).

Many choices of cell lines are suitable as the host cell for the presentinvention. Preferably, the cell is of a type in which the respectiveBoNT exhibits its toxic activities. In other words, the cells preferablydisplays suitable cell surface receptors, or otherwise allow the toxinto be translocated into the cell sufficiently efficiently, and allow thetoxin to cleave the suitable substrate polypeptide. Specific examplesinclude primary cultured neurons (cortical neuron, hippocampal neuron,spinal cord motor neuron, etc); PC12 cells or derived PC12 cell lines;primary cultured chromaphin cells; several cultured neuroblastoma celllines, such as murine cholinergic Neuro 2a cell line, human adrenergicSK-N-SH cell line, and NS-26 cell line. See e.g. Foster and Stringer(1999), Genetic Regulatory Elements Introduced Into Neural Stem andProgenitor Cell Populations, Brain Pathology 9: 547-567.

The coding region for the substrate-FRET polypeptide is under thecontrol of a suitable promoter. Depending on the types of host cellsused, many suitable promoters are known and readily available in theart. Such promoters can be inducible or constitutive. A constitutivepromoter may be selected to direct the expression of the desiredpolypeptide of the present invention. Such an expression construct mayprovide additional advantages since it circumvents the need to culturethe expression hosts on a medium containing an inducing substrate.Examples of suitable promoters would be LTR, SV40 and CMV in mammaliansystems; E. coli lac or trp in bacterial systems; baculovirus polyhedronpromoter (polh) in insect systems and other promoters that are known tocontrol expression in eukaryotic and prokaryotic cells or their viruses.Examples of strong constitutive and/or inducible promoters which arepreferred for use in fungal expression hosts are those which areobtainable from the fungal genes for xylanase (xlnA), phytase,ATP-synthetase, subunit 9 (oliC), triose phosphate isomerase (tpi),alcohol dehydrogenase (AdhA), alpha-amylase (amy), amyloglucosidase(AG—from the glaA gene), acetamidase (amdS) andglyceraldehyde-3-phosphate dehydrogenase (gpd) promoters. Examples ofstrong yeast promoters are those obtainable from the genes for alcoholdehydrogenase, lactase, 3-phosphoglycerate kinase and triosephosphateisomerase. Examples of strong bacterial promoters include SPO₂ promotersas well as promoters from extracellular protease genes.

Hybrid promoters may also be used to improve inducible regulation of theexpression construct. The promoter can additionally include features toensure or to increase expression in a suitable host. For example, thefeatures can be conserved regions such as a Pribnow Box or a TATA box.The promoter may even contain other sequences to affect (such as tomaintain, enhance or decrease) the levels of expression of thenucleotide sequence of the present invention. For example, suitableother sequences include the Shl-intron or an ADH intron. Other sequencesinclude inducible elements—such as temperature, chemical, light orstress inducible elements. Also, suitable elements to enhancetranscription or translation may be present. An example of the latterelement is the TMV 5′ signal sequence (see Sleat, 1987, Gene 217:217-225; and Dawson, 1993, Plant Mol. Biol. 23: 97).

The expression vector may also contain sequences which act on thepromoter to amplify expression. For example, the SV40, CMV, and polyomacis-acting elements (enhancer) and a selectable marker can provide aphenotypic trait for selection (e.g. dihydrofolate reductase or neomycinresistance for mammalian cells or ampicillin/tetracycline resistance forE. coli). Selection of the appropriate vector containing the appropriatepromoter and selection marker is well within the level of those skilledin the art.

Preferably the coding region for the substrate-FRET polypeptide is underthe control of an inducible promoter. In comparison to a constitutivepromoter, an inducible promoter is preferable because it allows forsuitable control of the concentration of the reporter in the cell,therefore the measurement of changes in FRET signals are greatlyfacilitated.

For example, FRET reporter can be controlled using the Tet-on & Tet-offsystem (BD Biosciences, San Jose, Calif.). Under the control of thispromoter, gene expression can be regulated in a precise, reversible andquantitative manner. Briefly, for Tet-on system, the transcription ofdownstream gene only happens when doxycycline is present in the culturemedium. After the transcription for a certain period of time, we canchange culture medium to deplete doxycycline, thus, stop the synthesisof new FRET reporter proteins. Therefore, there is no background fromnewly synthesized FRET proteins, and we may be able to see a fasterchange after toxin treatment.

Fluorescent analysis can be carried out using, for example, a photoncounting epifluorescent microscope system (containing the appropriatedichroic mirror and filters for monitoring fluorescent emission at theparticular range), a photon counting photomultiplier system or afluorometer. Excitation to initiate energy transfer can be carried outwith an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiberoptic light source, or other high intensity light source appropriatelyfiltered for excitation in the desired range. It will be apparent tothose skilled in the art that excitation/detection means can beaugmented by the incorporation of photomultiplier means to enhancedetection sensitivity. For example, the two photon cross correlationmethod may be used to achieve the detection on a single-molecule scale(see e.g. Kohl et al., Proc. Nat'l. Acad. Sci., 99:12161, 2002).

A number of parameters of fluorescence output may be measured. Theyinclude: 1) measuring fluorescence emitted at the emission wavelength ofthe acceptor (A) and donor (D) and determining the extent of energytransfer by the ratio of their emission amplitudes; 2) measuring thefluorescence lifetime of D; 3) measuring the rate of photobleaching ofD; 4) measuring the anisotropy of D and/or A; or 5) measuring the Stokesshift monomer/excimer fluorescence. See e.g. Mochizuki et al., (2001)“Spatio-temporal images of grow-factor-induced activation of Ras andRapl.” Nature 411:1065-1068, Sato et al. (2002) “Fluorescent indicatorsfor imaging protein phosphorylation in single living cells.” NatBiotechnol. 20:287-294.

In another embodiment, the present invention provides a method fordetecting BoNTs using surface plasmon resonance imaging (SPRi)techniques. Surface plasmon resonance (SPR) is an established opticaltechnique for the detection of molecular binding, based on thegeneration of surface plasmons in a thin metal film (typically gold)that supports the binding chemistry. Surface plasmons are collectiveoscillations of free electrons constrained in the metal film. Theseelectrons are excited resonantly by a light field incident from a highlyrefractive index prism. The angle of incidence q over which thisresonant excitation occurs is relatively narrow, and is characterized bya reduction in the intensity of the reflected light which has a minimumat the resonant angle of incidence qr. The phase of the reflected lightalso varies nearly linearly with respect to q in this region. The valueof qr is sensitive to the refractive index of the medium that resideswithin a few nanometers of the metal film. Small variations in therefractive index, due to the binding of a molecule to the film, or dueto the change in the molecular weight of the bound molecules, maytherefore be detected as a variation of this angle. Many methods areknown in the art for anchoring biomolecules to metal surfaces, fordetecting such anchoring and measuring SPRi are known in the art, seee.g. U.S. Pat. No. 6,127,129 U.S. Pat. No. 6,330,062, and Lee et al.,2001, Anal. Chem. 73: 5527-5531, Brockman et al., 1999, J. Am. Chem.Soc. 121: 8044-8051, and Brockman et al., 2000, Annu. Rev. Phys. Chem.51: 41-63, which are all incorporated herein by reference in theirentirety.

In practice a layer of BoNT target peptides may be deposited on themetal. A sample suspected of containing a corresponding BoNT is appliedto the composite surface, and incubated to allow the toxin, if present,to cleave the bound target peptides. The cleavage will result in thedecrease of the molecular weight of the peptide bound to the metalsurface, which decrease can then be detected using standard equipmentsand methods. A decrease in the thickness of the bound layer indicatesthat cleavage has occurred and in consequence, the sample contains thetoxin corresponding to the target peptide.

Alternatively, binding of a BoNT protein to its corresponding substratepeptide, which is anchored to the metal surface, will also cause achange in the refractive index and can be detected by known SPRitechniques and apparati.

Many methods are known in the art for anchoring or depositing protein orpeptides molecules on to a metal surface. For example, add an extra Cysresidue can be added to the end of the peptide, which can then becrosslinked onto the metal surface. Indirectly, an antibody can beanchored first to the metal surface, and to which antibody the toxinsubstrate can be bound. Indirect anchoring via antibodies is suitablefor the present invention so long as the antibody-substrate binding doesnot prevent the toxin from recognizing and accessing the cleavage siteof the substrate. Furthermore, nickel-NTA or glutathione that can beused to hold down his6 or GST fusion proteins, respectively. Additionalinformation regarding anchoring peptide to metal surface may be found inWegner et al, (2002) Characterization and Optimization of Peptide Arraysfor the Study of Epitope-Antibody Interactions Using Surface PlasmonResonance Imaging” Analytical Chemistry 74:5161-5168, which is alsoincorporated herein by reference in its entirety.

Changes of about 10-16 bases in a nucleic acid molecule, correspondingto 3,000 to 6,400 d in molecular weight, can be easily detected by SPR1.This implies that a change of as few as 16 amino acid residues in apeptide molecule can be detected. This high sensitivity allows theanchoring of a short peptide substrate onto the surface, instead ofusing the full-length toxin substrate proteins. Short peptide fragmentsare preferred because they are more stable, less expensive to prepareand allow higher reaction specificity.

EXAMPLES

Materials and Methods

Construction of bio-sensor DNA constructs: YFP cDNA (Clontech) wasinserted into the pECFP-C1 vector (Clontech) using EcoRI and BamHI siteto generate pECFP-YFP vector. cDNA encoding amino acid 33-94 of rat sybII was amplified using PCR and into pECFP-YFP vector using XhoI andEcoRI sites, which are between CFP and YFP gene, to generateCFP-SybII-YFP (also referred to as CFP-Syb (33-94)-YFP) construct thatcan be used to transfect cells. Construct CFP-SNAP-25-YFP (also referredto as CFP-SNAP-25 (141-206)-YFP) was built using the same method, butusing residues 141-206 of SNAP-25. A construct (CFP-SNAP25FL-YFP) withfull-length rat SNAP-25B as the linker was also made. In order to purifyrecombinant chimera proteins using bacteria E. coli, we also movedCFP-SybII-YFP gene and CFP-SNAP-25-YFP gene from pECFP-YFP vector into apTrc-his (Invitrogen) vector using NheI and BamHI sites.

The mutation of four Cys residues of SNAP-25 to Ala was accomplished bysite-directed mutagenesis using PCR, and the fragment was then insertedbetween CFP and YFP as described above.SNAP-25(83-120)-CFP-SNAP-25(141-206)-YFP were built by first insertingthe cDNA fragment that encoding the residues 83-120 of SNAP-25 into theXhoI/EcoRI sites of pEYFP-N1(Clontech), and then subcloningCFP-SNAP-25(141-206) cDNA into downstream sites using EcoRI/BamHI.YFP-Syb(FL)-CFP was built by first inserting a full length Syb II cDNAinto pECFP-C1 vectors at EcoRI and BamHI sites, and then inserting afull length YFP cDNA into the upstream at XhoI and EcoRI sites.YFP-Syb(33-116)-CFP was built by replacing full-length Syb inYFP-Syb(FL)-CFP construct via EcoRI/BamHI sites. All cDNA fragments weregenerated via PCR.

Protein purification and fluorescence spectra acquisition: His₆-taggedCFP-SybII-YFP and CFP-SNAP-25-YFP proteins were purified as described(Chapman et al., A novel function for the second C2 domain ofsynaptotagmin. Ca²⁺-triggered dimerization. J. Biol. Chem. 271,5844-5849 (1996)). Proteins were dialyzed using HEPES buffer (50 mMHEPES, pH 7.1) overnight. 300 nM protein was put into a cuvette in atotal volume of 500 μl HEPES buffer that contains 2 mM DTT and 10 μMZnCl₂. The emission spectra from 450 nM to 550 nM was collected using aPTIQM-1 fluorometer. The excitation wavelength is 434 nM, which is theoptimal excitation wavelength for CFP.

Activation of Botulinum neurotoxin and monitoring the cleavage ofbio-sensor proteins: BoNT/A, B, E or F was incubated with 2 mM DTT and10 μM ZnCl₂ for 30 min at 37° C. to reduce the toxin light chain fromthe heavy chain. For experiments using a PTIQM-1 fluorometer, 10 nMBoNT/A, E, or 50 nM BoNT/B, F were added into the cuvette that contains300 nM corresponding FRET sensors. The emission spectra were collectedas described above, at certain time intervals after adding toxins (e.g.0, 2, 5, 10, 30, 60, 90 min). At the end of each emission scan, a smallportion of the sample (30 μl) was collected, mixed with SDS-loadingbuffer, and later subjected onto a SDS-page gel. The sensor protein andthe cleavage products were visualized with an anti-his₆ antibody usingenhanced chemiluminescence (ECL) (Pierce).

For experiments using a spectrofluorometer, 300 nM FRET sensor proteinwere prepared in a 100 μl volume per well in a 96-well plate. Variousconcentrations of BoNTs were added into each well, and samples wereexcited at 434 nM. The emission spectra of YFP channel (527 nM), and CFPchannel (470 nM) were collected for 90 min at 30 sec interval. The FRETratio is determined by the ratio between YFP channel and CFP channel foreach data point.

Measure the FRET ratio change in live cells after toxin treatment DNAconstructs pECFP-SNAP25-YFP were used to transfect PC12 cells usingelectroporation (Bio-Rad). Cells were passed 24 hrs. after thetransfection, and 50 nM BoNT/A were added into the culture medium. Afterincubation for 72 hours with toxin, the fluorescence images of cellsthat express FRET sensor were collected using a Nikon TE-300 microscope.Two images of each cell (CFP channel and FRET channel) were collectedusing the following filter set (Chroma Inc.): CFP channel: CFPexcitation filter (436/10 nm), JP4 beamsplitter, CFP emission filter:(470/30 nm); FRET channel: CFP excitation filter (436/10 nm), JP4beamsplitter, YFP emission filter (535/30 nm). The background (the areasthat contain no cells) was subtracted from each image, and thefluorescence intensities of CFP channel and FRET channel of each cellwere compared using MetaMorph software. The FRET ratio is determined bythe intensity ratio between FRET channel and CFP channel as previousdescribed. Control cells were not treated with toxins but were analyzedin an identical manner. To test BoNT/B in live cells, we transfected aPC12 cell line that express syt II using the same procedure as describedabove.

Live cell imaging and FRET analysis: PC12 cells were transfected withvarious cDNA constructs indicated in the Figure legends viaelectroporation (Bio-Rad, CA). The fluorescence images were collectedusing a Nikon TE-300 microscope with a 100.times. oil-immersedobjective. CFP/YFP FRET in live cells was quantified using anestablished method with the three-filter set method (Gordon et al.,Quantitative fluorescence resonance energy transfer measurements usingfluorescence microscopy. Biophys J. 74, 2702-2713 (1998); Sorkin et al.,Interaction of EGF receptor and grb2 in living cells visualized byfluorescence resonance energy transfer (FRET) microscopy. Curr. Biol.10, 1395-1398 (2000)). In brief, three consecutive images were acquiredfor each cell, through three different filter sets: CFP filter(excitation, 436/10 nm; emission, 470/30 nm), FRET filter (excitation,436/10 nm; emission, 535/30 nm), and YFP filter (excitation, 500/20 nm,emission, 535/30 nm). A JP4 beam splitter (Set ID 86000, Chroma Inc. VT)was used. All images were acquired with exact the same settings(4.times.4 Binning, 200 ms exposure time). In order to exclude theconcentration-dependent FRET signal that can arise from high expressionlevel of fluorescence proteins, only cells with CFP and YFP intensitiesbelow the half value of the maximal 12-bit scale (1-2097 gray scale)were counted in our experiments (Miyawaki et al., Monitoring proteinconformations and interactions by fluorescence resonance energy transferbetween mutants of green fluorescent protein. Methods Enzymol. 327,472-500 (2000); Erickson et al., DsRed as a potential FRET partner withCFP and GFP. Biophys J 85, 599-611 (2003)). The background (from areasthat did not contain cells) was subtracted from each raw image beforeFRET values were calculated. The fluorescence intensity values of eachimage were then obtained and compared. PC12 cells transfected with CFPor YFP alone were first tested in order to obtain the crosstalk valuefor these filter sets. The FRET filter channel exhibits about 56-64% ofbleed-through for CFP, and about 24% for YFP. There is virtually nocrosstalk for YFP while using the CFP filter, or for CFP while using theYFP filter, which greatly simplified the FRET calculations. For cellsexpressing toxin sensors, the “corrected FRET” value was calculatedusing the following equation: correctedFRET=FRET−(CFP.times.0.60)−(YFP.times.0.24), where FRET, CFP and YFPcorrespond to fluorescence intensity of images acquired through FRET,CFP and YFP filter sets, respectively. The average fraction ofbleed-through coming from CFP and YFP fluorescence are 0.6 and 0.24,respectively, when acquiring image through the FRET filter set. Becausetoxin cleavage of the CFP-SNAP25FL-YFP sensor resulted in the membranedissociation of YFP fragment, which was degraded in the cytosol (FIG. 7panel C, panel E), the FRET ratio used in our data analysis iscalculated as normalizing “corrected FRET” value to only the CFPfluorescence intensity (corrected FRET/CFP). We note that the CFPintensity in these calculations was an underestimate due to donorquenching if FRET occurred. However, it has been reported the decreasein CFP fluorescence because of donor quenching is only about 5-10%(Gordon et al., Quantitative fluorescence resonance energy transfermeasurements using fluorescence microscopy. Biophys J 74, 2702-2713(1998); Sorkina et al., Oligomerization of dopamine transportersvisualized in living cells by fluorescence resonance energy transfermicroscopy. J. Biol. Chem. 278, 28274-28283 (2003)). All images andcalculations were performed using MetaMorph software (Universal ImagingCorp., PA).

For experiments involving toxin treatment, indicated holotoxins wereadded to the cell culture media for various time, and cells were thenanalyzed as described above. Control cells were transfected with toxinsensors but not treated with toxins, and they were analyzed in anidentical manner.

Immunoblot analysis of toxin substrate cleavage: Wild type PC12 cells orSyt II+PC12 cells (Dong et al., 2003, supra) were transfected withvarious toxin sensor cDNA constructs as indicated in the Figure legends.BoNT/A or B was added to the culture medium 24 h after transfection andcells were incubated for another 48 hrs. Cells were then harvested andcell lysates were subject to immunoblot analysis as describedpreviously. Control cells were transfected with the same cDNA constructsand assayed in parallel except they were not treated with toxins. Onethird of the control cell lysates were treated with toxins in vitro (200nM BoNT/A or B, 30 min at 37° C.), and subjected to immunoblot analysis.Endogenous SNAP-25 and transfected CFP-SNAP-25-YFP sensors were assayedusing an anti-SNAP-25 antibody 26. CFP-SNAP-25-YFP and CFP-SybII-YFPsensor proteins were also assayed using a GFP polyclonal antibody (SantaCruz, Calif.). An anti-his6 antibody (Qigen Inc., CA) was used to assayfor recombinant sensor protein cleavage.

Example 1: Bio-Sensors Based on CFP-YFP FRET Pair and BotulinumNeurotoxin Protease Activity

In order to monitor botulinum neurotoxin protease activity using FRETmethod, CFP and YFP protein are connected via syb II or SNAP-25fragment, denoted as CFP-SybII-YFP and CFP-SNAP-25-YFP, respectively(FIG. 1 panel A). Short fragments of toxin substrates were used insteadof the full-length protein to optimize the CFP-YFP energy transferefficiency, which falls exponentially as the distance increases.However, the cleavage efficiency by BoNTs decreases significantly as thetarget protein fragments get too short. Therefore, the region thatcontain amino acid 33-96 of synaptobrevin sequence was used because ithas been reported to retain the same cleavage rate by BoNT/B, F, andTeNT as the full length synaptobrevin protein does. Similarly, residues141-206 of SNAP-25 were selected to ensure that the construct can stillbe recognized and cleaved by BoNT/A and E.

The FRET assay is depicted in FIG. 1 panel B. When excited at 434 nM(optimal excitation wavelength for CFP), the CFP-SybII-YFP andCFP-SNAP-25-YFP chimera protein would elicit YFP fluorescence emissionbecause of the FRET between CFP-YFP pair. Botulinum neurotoxins canrecognize and cleave the short substrate fragments between CFP and YFP,and FRET signal will be abolished after CFP and YFP are separated.Because these chimera proteins can be expressed in living cells, theyare also denoted as “bio-sensor” for botulinum neurotoxins.

We first purified his₆-tagged recombinant chimera protein ofCFP-SybII-YFP, and CFP-SNAP-25-YFP, and characterized their emissionspectra using a PTIQM-1 fluorometer. As expected, both bio-sensorproteins show an obvious YFP fluorescence peak at 525 nM when their CFPwere excited at 434 nM (FIG. 2 panel B, panel C). On the contrary, theYFP alone only gave small fluorescence signal when excited directly at434 nM (FIG. 2 panel A). The mixture of individual CFP and YFP doesn'thave the peak emission at 525 nM (FIG. 2 panel A). This demonstrated theYFP fluorescence peak observed using bio-sensor proteins resulted fromFRET. Because the FRET ratio (YFP fluorescence intensity/CFPfluorescence intensity) was affected by many factors, such as buffercomposition, the Zn²⁺ concentration and the concentration of reducingagents (data not shown), the experiments thereafter were all carried outin the same buffer conditions (50 mM HEPES, 2 mM DTT, 10 μM ZnCl₂, pH7.1). 2 mM DTT and 10 μM Zn²⁺ were added to optimize the botulinumneurotoxin protease activity.

Example 2: Monitoring the Cleavage of Bio-Sensor Proteins by BotulinumNeurotoxins In Vitro

300 nM chimera protein CFP-SybII-YFP was mixed with 50 nM pre-reducedBoNT/B holotoxin in a cuvette. The emission spectra were collected atdifferent time points after adding BoNT/B (0, 2, 5, 10, 30, 60 min,etc). At the end of each scan, a small volume of sample (30 μl) wastaken out from the cuvette and mixed with SDS-loading buffer. Thesesamples later were subjected to SDS-page gels and the cleavage ofchimera proteins were visualized using an antibody against the his₆ tagin the recombinant chimera protein. As shown in FIG. 3 panel A, theincubation of bio-sensor protein with BoNT/B resulted in a decrease ofYFP emission and increase of CFP emission. The decrease of FRET ratio isconsistent with the degree of cleavage of the chimera protein by BoNT/B(FIG. 3 panel A, lower portion). This result demonstrates the cleavageof the bio-sensor protein can be monitored in real time by recording thechange in its FRET ratio.

The same assay was performed to detect CFP-SybII-YFP cleavage by BoNT/F,and CFP-SNAP-25-YFP cleavage by BoNT/A or E (FIG. 3 panel B, panel C,panel D). Similar results were obtained with the experiment usingBoNT/B. IN all cases, we observed the same kinetics of cleavage of thesubstrate using both the optical readout and the immunoblot blotanalysis. BoNT/A and E cleaved their chimera substrate much faster thanBoNT/B and F did in our assay. Thus, only 10 nM BoNT/A or E were used inorder to record the change occurred within first several minutes. Thecleavage of chimera protein is specific, since mixing BoNT/B and F withCFP-SNAP-25-YFP, or mixing BoNT/A and E with CFP-SybII-YFP did notresult in any change in FRET ratio or substrate cleavage (data notshown).

Example 3: Monitoring Botulinum Neurotoxin Protease Activity in RealTime Using a Microplate Spectrofluorometer

The above experiments demonstrated that the activity of botulinumneurotoxin can be detected in vitro by monitoring the changes of theemission spectra of their target bio-sensor proteins. We then determinedif we could monitor the cleavage of bio-sensor proteins in real timeusing a microplate reader—this will demonstrate the feasibility to adaptthis assay for future high-throughput screening. As shown in FIG. 4panel A, 300 nM CFP-SNAP-25-YFP chimera protein was mixed with 10 nMBoNT/A in a 96-well plate. CFP was excited at 436 nm and thefluorescence of the CFP channel (470 nM) and YFP channel (527 nM) wererecorded over 90 min at 30 sec intervals. Addition of BoNT/A resulted inthe decrease of YFP channel emission and the increase of CFP channelemission. This result enabled us to trace the kinetics of botulinumneurotoxin enzymatic activity in multiple samples in real time. Forinstance, as shown in FIG. 4 panel B, various concentration of BoNT/A orE were added into 300 nM CFP-SNAP-25-YFP, and the FRET ration of eachsample were monitored simultaneously as described in FIG. 4 panel A.Changes in the FRET ratio were related to the toxin concentration—highertoxin concentration resulted in faster decrease of the FRET ratio. Thischange in FRET ratio is specific, because no significant change wasdetected for either CFP-SNAP-25-YFP alone (FIG. 4 panel C left side) ora mixture of CFP and YFP (FIG. 4 panel C right side).

Although it would be difficult to correlate the FRET ratio change withthe actual cleavage of the bio-sensor proteins at this stage, thismethod still provides the easiest way to compare toxin cleavage kineticsamong multiple samples when these samples were prepared and scannedsimultaneously—it is particularly useful for high throughput screeningtoxin inhibitors because it would provide information about how theinhibitor affects toxin enzymatic activities. We note that the unit foreach kinetic parameter would be the FRET ratio instead of substratesconcentration in these cases.

The sensitivity of this FRET based assay is determined by incubatingvarious concentrations of toxins with fixed amount of their targetbio-sensor proteins for certain period of time. The FRET ratio isrecorded using a microplate spectro-fluorometer, and plotted againsttoxin concentration. As shown in FIG. 5 panel A, this method has similarsensitivities for BoNT/A and E after 4 hours incubation (EC50 for BoNT/Ais 15 pM, and for BoNT/E is 20 pM, upper panel), and incubation for 16hours slightly increased the detection sensitivity (FIG. 5 panel A,lower portion). The sensitivities for BoNT/B and F are close to eachother, but are about 10 times lower than BoNT/A and E with 4 hoursincubation (FIG. 5 panel B, upper portion), EC50 is 242 pM for BoNT/B,and 207 pM for BoNT/F). Extension of the incubation period to 16 hoursincreased the ability to detect BoNT/B and BoNT/F activity by 8-fold and2-fold, respectively.

Example 4: Monitoring Botulinum Neurotoxin Activity in Live Cells

CFP-YFP based bio-sensor assay not only can be used to detect botulinumneurotoxin in vitro, but also can be used in live cells. To establishthis application, PC 12 cells were transfected with CFP-SNAP-25-YFP.PC12 cell is a neuroendocrine cell line that is able to take up BoNT/Aand E. Transfected cells were incubated with BoNT/A (50 nM) for 72hours, and the FRET ratio of cells that express CFP-SNAP25-YFP wererecorded using a epi-fluorescence microscope equipped with specialfilter sets for CFP-YFP FRET detection. Briefly, the FRET ratio iscalculated as the ratio between the fluorescence intensity of the imagesfrom the same cell collected using two filter sets, one for CFP(excitation 437 nm/emission 470 nm), and another for FRET (excitation437 nm/emission 535 nm). A total number of 53 cells were collected, andcompared to the same number of control cells which express the samebio-sensor protein but were not exposed to toxin. As shown in FIG. 6panel A, BoNT/A treatment for 72 hours significantly decreased FRETratio for the cell population that was examined (p<1.47E-05). Wild typePC12 cells are not sensitive to BoNT/B and F.

A PC12 cell line was recently created that expresses both synaptotagminII, a receptor for BoNT/B, and CFP-SybII-YFP bio-sensor. These cellswere used to detect BoNT/B action in live cells. As shown in FIG. 6panel B, BoNT/B (30 nM) treatment for 72 hours significantly decreasedFRET ratio of the bio-sensor proteins expressed in cells (p<2.1E-10). Wenote that there were still large number of cells that do not appear tochange FRET ratio for both bio-sensor proteins. There are severalpossible explanations. First, the toxin/bio-sensor protein ratio may betoo low in these cells, thus, the significant cleavage of bio-sensorproteins may require a longer incubation time. Second, these cells mayhave high level of protein synthesis activity, thus new bio-sensorprotein was synthesis quickly to replace cleavage products.Nevertheless, these experiments demonstrate the feasibility to adoptthis FRET based assay in living cells and neurons.

Example 5: Cell Based Detection of BoNTs

To carry out cell-based studies, we first transfected PC12 cells withCFP-SNAP-25(141-206)-YFP sensor (FIG. 7 panel A). The FRET signal inliving cells was acquired using an established three-filter set methodwith an epi-fluorescence microscope as shown in FIG. 2 panel A (Gordon,et al., Quantitative fluorescence resonance energy transfer measurementsusing fluorescence microscopy. Biophys. J. 74, 2702-2713 (1998); andSorkin et al., Interaction of EGF receptor and grb2 in living cellsvisualized by fluorescence resonance energy transfer (FRET) microscopy.Curr. Biol. 10, 1395-1398 (2000), as described above in the Materialsand Methods Section. Transfected PC12 cells were treated with 50 nMBoNT/A for 96 hrs. Their fluorescence images were analyzed and thenormalized FRET ratio (corrected FRET/CFP) was plotted in FIG. 7 panelB. Although SNAP-25(141-206) fragments were reported to have similartoxin cleavage rates as full length SNAP-25 in vitro (Washbourne et al.,Botulinum neurotoxin types A and E require the SNARE motif in SNAP-25for proteolysis. FEBS Lett. 418, 1-5 (1997)), CFP-SNAP-25(141-206)-YFPappeared to be a poor toxin substrate in living cells. Incubation ofBoNT/A (50 nM) for 96 hr exhibited a small (but significant) shift inthe FRET ratio among the cell population, indicating the cleavage isinefficient in cells, and this sensor is not practical for toxindetection in cells.

Surprisingly, we found that using full length SNAP-25 as the linkerbetween CFP and YFP yielded significant levels of FRET when expressed inPC12 cells, despite the fact that SNAP-25 is 206 amino acid residueslong (FIG. 7 panel C, and FIG. 8 panel C). This FRET signal is dependenton the membrane anchor of SNAP-25 since mutation of the palmitoylationsites within SNAP-25 (Cys 85, 88, 90, 92 Ala) (Lane & Liu,Characterization of the palmitoylation domain of SNAP-25. J. Neurochem.69, 1864-1869 (1997); Gonzalo et al., SNAP-25 is targeted to the plasmamembrane through a novel membrane-binding domain. J. Biol. Chem. 274,21313-21318 (1999); Koticha et al., Plasma membrane targeting of SNAP-25increases its local concentration and is necessary for SNARE complexformation and regulated exocytosis. J. Cell Sci. 115, 3341-3351 (2002);Gonelle-Gispert et al., Membrane localization and biological activity ofSNAP-25 cysteine mutants in insulin-secreting cells. J. Cell Sci. 113(Pt 18), 3197-3205 (2000)), which results in the cytosolic distributionof the protein (denoted as CFP-SNAP-25(Cys-Ala)-YFP), significantlyreduced the FRET signal (FIG. 7 panel D). This finding suggests themembrane anchoring of SNAP-25 may result in conformational changes thatbring N-terminus and C-terminus of SNAP-25 close to each other. Thissensor is denoted as CFP-SNAP-25(FL)-YFP. Incubation of cells thatexpress CFP-SNAP-25(FL)-YFP with 50 nM BoNT/A resulted in a progressivedecrease in the FRET ratio over time (FIG. 7 panel C, right side).BoNT/A cleavage of the sensor resulted in two fragments: an N-terminalfragment of SNAP-25 tagged with CFP that remained on the membrane (FIG.2 panel C, middle portion), and a short C-terminal fragment of SNAP-25tagged with YFP that was expected to redistribute into the cytosol aftertoxin cleavage. Interestingly, we noticed the C-terminal cleavageproduct, SNAP-25(198-206)-YFP, largely disappeared after the toxincleavage (FIG. 2 panel C, the “YFP” micrograph in the middle portion).This observation was confirmed by immunoblot analysis (FIG. 7 panel E),indicating this soluble fragment was degraded much faster than the otherfragment that is retained on the membrane. This unexpected resultprovides an alternative way to detect toxin activity in living cells bysimply monitoring the ratio between CFP and YFP fluorescence.

It was recently reported that the BoNT/A light chain contains membranelocalization signals and targets to the plasma membrane indifferentiated PC12 cells (Fernandez-Salas et al., Plasma membranelocalization signals in the light chain of botulinum neurotoxin. Proc.Natl. Acad. Sci. USA 101, 3208-3213 (2004). Thus, we investigated thepossibility that membrane anchoring of SNAP-25 is critical for efficientcleavage by BoNT/A. To directly test this idea,CFP-SNAP-25(Cys-Ala)-YFP, which was distributed in the cytosol (FIG. 7panel D), and CFP-SNAP-25(FL)-YFP, which was anchored to the plasmamembrane, were used to transfect PC12 cells and assayed in parallel forthe cleavage by BoNT/A in cells. As indicated in FIG. 7 panel E,incubation of cells with BoNT/A resulted in the cleavage of significantamount of CFP-SNAP-25(FL)-YFP sensor, while there was no detectablecleavage of CFP-SNAP-25 (Cys-Ala)-YFP under the same assay conditions.

To further confirm this finding, we also targeted the inefficient sensorthat contains SNAP-25(141-206) to the plasma membrane using a shortfragment of SNAP-25 (residues 83-120, which can target GFP to plasmamembranes (Gonzalo et al., SNAP-25 is targeted to the plasma membranethrough a novel membrane-binding domain. J. Biol. Chem. 274, 21313-21318(1999)) (FIG. 8 panel A). As expected, anchoring of theCFP-SNAP-25(141-206)-YFP sensor to the plasma membrane resulted inefficient cleavage by BoNT/A (FIG. 8 panel B). These findings indicatethat the sub-cellular localization of SNAP-25 is indeed critical for theefficient cleavage by BoNT/A in cells.

We next tested whether the CFP-Syb(33-94)-YFP sensor could be used toassay BoNT/B activity in cells. For these studies, we used a PC12 cellline that expresses synaptotagmin II, which mediates BoNT/B entry intocells (Dong et al. Synaptotagmins I and II mediate entry of botulinumneurotoxin B into cells. J. Cell Biol. 162, 1293-1303 (2003)). As shownin FIG. 9 panel A (upper portion), transfected CFP-Syb(33-94)-YFP is asoluble protein present throughout the cells. Similar to the case ofCFP-SNAP-25(141-206)-YFP, BoNT/B (50 nM, 96 hr) treatment only slightlydecreased the FRET ratio (FIG. 9 panel A, lower portion), indicatingthat the cleavage of this sensor is also inefficient in cells.

Our experience with the BoNT/A sensor prompted us to investigate whetherthe sub-cellular localization of Syb is important for cleavage of Syb byBoNT/B. Endogenous Syb is 116 amino acid residues long, and resides onsecretory vesicles through a single transmembrane domain (residues95-116, FIG. 9 panel B). In order to ensure proper vesicularlocalization, we used full-length Syb as the linker between CFP and YFP.Because CFP is relatively resistant to the acidic environment in thevesicle lumen (Tsien, The green fluorescent protein. Annu. Rev Biochem67, 509-544 (1998), the CFP was fused to the C-terminus of Syb and ispredicted to reside inside vesicles, while the YFP was fused to theN-terminus of Syb and faces the cytosol (FIG. 9 panel B). This sensor isdenoted as YFP-Syb(FL)-CFP. Since FRET is unlikely to occur between CFPand YFP here, a novel approach was taken to monitor the cleavage byBoNT/B. Cleavage of YFP-Syb(FL)-CFP sensor would generate two fragments,including a N-terminal portion tagged with YFP, which would be releasedinto the cytosol, and a short C-terminal portion tagged with CFP that isrestricted inside vesicles. Thus, toxin activity would result in theredistribution of YFP fluorescence in cells. YFP-Syb(FL)-CFP proved tobe an efficient toxin sensor in cells. As indicated in FIG. 9 panel C,treatment with BoNT/B resulted in the dissociation of YFP from thevesicles and its redistribution into the cytosol. We note that thesoluble YFP fragment was able to enter the nucleus, where there was nofluorescence signal prior to toxin treatment (FIG. 9 panel C), providingan area where the YFP redistribution can be readily detected. Unlike theFRET assay, this detection method does not require a short distancebetween CFP and YFP, thus providing a novel approach to monitor proteaseactivity in living cells.

To exclude the possibility that the inefficient cleavage of the sensorcontaining Syb(33-94) fragment is due to the lack of the N-terminal 32amino acid, a sensor containing the truncated form of Syb that lacks theN-terminal 32 residues (denoted as CFP-Syb(33-116)-YFP) was built. Thissensor contains the same cytosolic domain of Syb with the inefficientsensor (residues 33-94), plus the transmembrane domain (residues95-116), which anchors it to vesicles. When assayed in parallel,significant amount of CFP-Syb(33-116)-YFP was cleaved by BoNT/B after 48hours, while there was no detectable cleavage of CFP-Syb(33-94)-YFP(FIG. 9 panel D), indicating the vesicular localization determines thecleavage efficiency in cells. This conclusion is further supported by arecent report that the presence of negatively charged lipid mixturesenhanced the cleavage rate of Syb by BoNT/B, TeNT, and BoNT/F in vitro(Caccin et al., VAMP/synaptobrevin cleavage by tetanus and botulinumneurotoxins is strongly enhanced by acidic liposomes. FEBS Lett. 542,132-136 (2003). It is possible that toxins may favor binding tovesicular membranes in cells, thus increasing the chance to encounterSyb localized on vesicles. Alternatively, it is also possible that thepresence of the transmembrane domain may be critical for maintaining aproper conformational state of Syb that is required for efficientcleavage.

Using full length SNAP-25 and Syb II as the linkers provided excellentoptical reporters that can mirror endogenous substrate cleavage inliving cells. These two reporters should be able to detect all sevenbotulinum neurotoxins and tetanus neurotoxin (TeNT). The substratelinker sequence can be readily modified to achieve specific detectionfor individual BoNTs or TeNT by changing the length or mutating othertoxin cleavage or recognition sites. These toxin biosensors shouldenable the cell-based screening of toxin inhibitors, and the study oftoxin substrate recognition and cleavage in cells.

The foregoing description and examples have been set forth merely toillustrate the invention and are not intended to be limiting. Sincemodifications of the disclosed embodiments incorporating the spirit andsubstance of the invention may occur to persons skilled in the art, theinvention should be construed broadly to include all variations fallingwithin the scope of the appended claims and equivalents thereof. Allreferences cited hereinabove and/or listed below are hereby expresslyincorporated by reference.

It should be apparent to those skilled in the art that many moremodifications besides those already described are possible withoutdeparting from the inventive concepts herein. The inventive subjectmatter, therefore, is not to be restricted except in the spirit of theappended claims. Moreover, in interpreting both the specification andthe claims, all terms should be interpreted in the broadest possiblemanner consistent with the context. In particular, the terms “comprises”and “comprising” should be interpreted as referring to elements,components, or steps in a non-exclusive manner, indicating that thereferenced elements, components, or steps may be present, or utilized,or combined with other elements, components, or steps that are notexpressly referenced. Where the specification claims refers to at leastone of something selected from the group consisting of A, B, C . . . andN, the text should be interpreted as requiring only one element from thegroup, not A plus N, or B plus N, etc.

What is claimed is:
 1. A method of screening for an inhibitor of abotulinum neurotoxin, comprising: providing a cell geneticallyengineered to express a construct comprising donor label and an acceptorlabel positioned to provide an electronic coupling such that the donorcan transfer energy to the acceptor by a dipole-dipole couplingmechanism, and a linker disposed between the donor label and theacceptor label, wherein the linker is a peptide sequence comprising (a)a cleavage site peptide comprising a SNARE motif, (b) a first peptidespacer interposed between the donor and the cleavage site peptide, and(c) a second peptide spacer interposed between the acceptor and thecleavage site peptide, wherein the first peptide spacer and the secondpeptide spacer each comprise three to fifteen amino acids and areselected to both increase the primary structure distance between thedonor and the acceptor and reduce the tertiary structure distancebetween the donor and the acceptor relative to a corresponding constructlacking the first spacer and second spacer, such that the construct ischaracterized by increased electronic coupling between the donor and theacceptor relative to a corresponding construct lacking the first spacerand second spacer; characterizing a first signal from the cell;contacting the cell with a botulinum neurotoxin in the presence of acandidate inhibitor; and following contacting the cell with thebotulinum neurotoxin, characterizing a second signal from the cell. 2.The method of claim 1, comprising the step of identifying the candidateinhibitor as an effective inhibitor of the botulinum neurotoxin whencomparison of the second signal to the first signal is indicative of alack of decrease in the electronic coupling.
 3. The method of claim 1,wherein the candidate compound is a member of a library of compounds. 4.The method of claim 1, wherein the method is a high throughput method.5. The method of claim 1, wherein at least one of the donor and theacceptor is at least one of a fluorophore and a chromophore.
 6. Themethod of claim 1, wherein the dipole-dipole coupling mechanism is aFörster resonance energy transfer (FRET).
 7. The method of claim 1,wherein the linker has a primary structure length≥5 nm.
 8. The method ofclaim 1, wherein the linker has a primary structure length≥8 nm.
 9. Themethod of claim 1, wherein the linker has a primary structure length≥12nm.
 10. The method of claim 1, wherein the cleavage site peptidecomprises a SNARE protein mutein.
 11. The method of claim 1, wherein thecandidate inhibitor comprises an activity related to cellular entry tothe cell.
 12. The method of claim 1, wherein the candidate inhibitorcomprises an activity related to translocation through a vesiclemembrane of the cell.
 13. The method of claim 1, wherein the candidateinhibitor interacts with a receptor of the genetically engineered cell.14. A method of screening for an inhibitor of a botulinum neurotoxin,comprising: providing a cell genetically engineered to express aconstruct comprising donor label and an acceptor label positioned toprovide an electronic coupling such that the donor can transfer energyto the acceptor by an electronic hop between the donor label and theacceptor label, and a linker disposed between the donor label and theacceptor label, wherein the linker is a flexible peptide sequencecomprising (a) a cleavage site peptide comprising a SNARE motif, (b) afirst peptide spacer interposed between the donor and the cleavage sitepeptide, and (c) a second peptide spacer interposed between the acceptorand the cleavage site peptide, wherein the first peptide spacer and thesecond peptide spacer each comprise three to fifteen amino acids and areselected to both increase the primary structure distance between thedonor and the acceptor and reduce the tertiary structure distancebetween the donor and the acceptor relative to a corresponding constructlacking the first spacer and second spacer, such that the construct ischaracterized by an increase in the electronic hop between the donor andthe acceptor relative to a corresponding construct lacking the firstspacer and second spacer; characterizing a first signal from the cell;contacting the cell with a botulinum neurotoxin in the presence of acandidate inhibitor; and following contacting the cell with thebotulinum neurotoxin, characterizing a second signal from the cell. 15.The method of claim 13, comprising the step of identifying the candidateinhibitor as an effective inhibitor of the botulinum neurotoxin whencomparison of the second signal to the first signal is indicative of alack of decrease in the electronic coupling.
 16. The method of claim 14,wherein the candidate compound is a member of a library of compounds.17. The method of claim 14, wherein the method is a high throughputmethod.
 18. The method of claim 14, wherein at least one of the donorand the acceptor is at least one of a fluorophore and a chromophore. 19.The method of claim 14, wherein the linker has a primary structurelength≥5 nm.
 20. The method of claim 14, wherein the linker has aprimary structure length≥8 nm.
 21. The method of claim 14, wherein thelinker has a primary structure length≥12 nm.
 22. The method of claim 14,wherein the cleavage site peptide comprises a SNARE protein mutein. 23.The method of claim 14, wherein the candidate inhibitor comprises anactivity related to cellular entry to the cell.
 24. The method of claim14, wherein the candidate inhibitor comprises an activity related totranslocation through a vesicle membrane of the cell.
 25. The method ofclaim 14, wherein the candidate inhibitor interacts with a receptor ofthe genetically engineered cell.